Project Summary Cancer metastasis accounts for approximately 90% of cancer-associated deaths, yet the mechanisms underlying this process remain the least understood aspect of cancer biology. During metastasis, tumor cells spread from the primary tumor to surrounding tissues and to distant organs. The most intriguing aspect of cancer metastasis is that despite the epithelial origin of most primary tumors, these tumor cells gain the ability to detach, invade and migrate, such that they can escape their birth place and reach a distant site. What induces such dramatic changes in the primary tumor cells? An interesting possibility is that cancer cells may acquire their migratory potential by fusing with migratory cells, such that the many genes expressed by the latter can be hijacked by cancer cells to promote their migration. Such cancer cell fusion hypothesis has been around for over a century, and the migratory cells are proposed to be leukocytes, such as macrophages. Indeed, various types of cancer cells have been shown to fuse with macrophages in vitro. Moreover, hybrid cells with biomarkers of both cancer cells and leukocytes have been observed in human cancer biopsies and peripheral blood. However, the percentage of the cancer cell-macrophage hybrids indicated by double positive biomarkers is relatively low (1- 20%), raising questions about the physiological relevance of cancer cell-macrophage fusion (CMF) in cancer metastasis. In addition, no one has observed CMF at the primary tumor site, and the molecular mechanism underlying such fusion remains completely unknown. Therefore, the CMF model has gained little traction in the metastasis field to date. Despite these uncertainties, the presence of CMF in various metastatic cancers, albeit detected at a low level, makes the model appealing nevertheless. Moreover, it is well known that the intrinsically fusion-prone macrophages can infiltrate the TME and settle right next to cancer cells, making CMF physically possible. I propose to detect the presence of cancer cell-macrophage hybrid using single cell RNAseq, visualize cancer cell-macrophage fusion using live cell imaging, and identify the molecular components mediating cancer cell-macrophage fusion. If definitive evidence can be obtained to support the CMF model, this will bring about a paradigm shift in our understanding of cancer metastasis and provide basis for novel therapeutic approaches for cancer treatment.